Adult spinal opioid receptor μ1 expression after incision is altered by early life repetitive tactile and noxious procedures in rats

Clinical and experimental data suggests that noxious stimulation at critical stages of development results in long‐term changes on nociceptive processing in later life. Here, we use an established, well‐documented rat model of repetitive noxious procedures closely mimicking the clinical situation in the NICU. In order to understand molecular changes underlying the long‐term consequences of repetitive stimulation of the developing nociceptive system the present study aims to analyze the presence of the µ‐opioid‐receptor‐1 (OPRM1). Neonatal rats received either four needle pricks per day in the left hind‐paw from postnatal day 0–7 as a model of procedural pain in infancy. Control pups were handled in the same way but were instead tactile stimulated, or were left undisturbed. At the age of 8 weeks, all animals received an ipsilateral hind‐paw incision as a model for post‐operative pain, and mechanical sensitivity was tested at multiple time‐points. Before, and 1 or 5 days post‐incision, spinal cord tissue was collected for immunostaining of opioid receptor OPRM1. Semi‐quantitative immunocytochemical analysis of superficial laminae in lumbar spinal dorsal horn revealed that: (1) early life repetitive tactile or noxious procedures do not alter baseline levels of OPRM1 staining intensity and (2) early life repetitive tactile or noxious procedures lead to a decrease in OPRM1 staining intensity 5 days after incision in adulthood compared to undisturbed controls. We conclude that early life repetitive tactile or noxious procedures affect the intensity of OPRM1‐immunoreactivity in the lumbar superficial spinal cord dorsal horn after adulthood injury, without affecting baseline intensity. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 78: 417–426, 2018     

Apolipoprotein A-I expression in rats is not altered by troglitazone

Insulin is known to upregulate apolipoprotein A-I (apoA-I) promoter activity and to increase apoA1 gene expression in vivo. To determine if enhancement of insulin action with insulin sensitizers can also increase the apoA-I expression, we studied the in vivo effect of troglitazone, a potent insulin sensitizer, on the expression of rat hepatic and intestinal apoA-I mRNA using Northern blot analysis. The plasma, hepatic, and intestinal apoA-I content was also measured with immunoblot analysis using a specific anti-rat apoA-I antiserum. Troglitazone, given mixed with rat chow (0.2%) for 18 days, did not increase either plasma or tissue apoA-I mRNA or protein content. Intestinal apoA-I mRNA content relative to glyceraldehyde-3 phosphate dehydrogenase (G(3)PDH) mRNA was significantly lower compared with hepatic tissue content in both control and troglitazone-treated rats. The effect of troglitazone on the rat apoA-I promoter was examined using transient transfection analysis in HepG2 cells transfected with the apoA-I-chloramphenicol acetyl transferase (CAT) reporter plasmid (pAI.474.CAT). CAT activity (percentage acetylation of chloramphenicol as means +/- SEM) was not significantly different in ethanol (vehicle)-treated cells compared with cells treated with troglitazone (50.5% +/- 2.5% in control cells vs 57.7% +/- 8.2% and 53.5% +/- 4.2% in cells treated with 10 and 100 mM troglitazone, respectively). It is concluded that troglitazone doses known to achieve insulin sensitization did not enhance rat apoA-I promoter activity sufficiently to result in an increased apoA-I mRNA or protein expression in the intact rat. However, peroxisome proliferator activator receptor (PPAR) agonists that have significant PPAR alpha activity in addition to their PPAR gamma effects, may well be able to induce apoA-I expression.     

Protease nexin-1 expression is altered in human breast cancer

Background  

Urokinase-type Plasminogen Activator (uPA), a serine protease, plays a pivotal role in human breast cancer metastasis by mediating the degradation of extracellular matrix proteins and promoting cell motility. In more advanced breast cancers, uPA activity is significantly up regulated and serves as a prognostic indicator of poor patient outcome. Classically, regulation of uPA activity, especially in breast cancers, is thought to be mediated by Type 1 Plasminogen Activator Inhibitor (PAI-1). However, we have recently found that a lesser known natural inhibitor of uPA, Protease Nexin 1 (PN-1), is expressed in normal human mammary tissue. Based on this observation, we investigated if PN-1 is also expressed in human breast cancers where it may contribute to the regulation of uPA and participate in the development of a metastatic phenotype.     

Alterations in gene expressions encoding preproET-1 and NOS in pulmonary tissue in endotoxemic rats

Septic shock is characterized by hypotension and a hyporeactive response to vasopressor agents. The pathogenesis is due to vascular leaks and an increased synthesis of cytokines and nitric oxide (NO). The present study examined the time-dependent alterations of endothelin-1 (ET-1) and the expression of NO synthase (NOS) in lung tissue in a septic rat model. Normal Sprague-Dawley (SD) rats aged 10 weeks received 15 mg/kg lipopolysaccharide (LPS) and then were sacrificed at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. Both systolic and diastolic pressure decreased in SD rats after LPS administration. Time-dependent onset of features of acute lung injury, such as the infiltration of inflammatory cells and thickening of alveolar septa, were seen in rats that received LPS. A 2.8-fold increase in the expression of preproET-1 level was observed in lung tissue 6 hrs after LPS administration. The expression of endothelial NOS (eNOS) was also altered in lung tissue in a time-dependent fashion. After the administration of LPS, there was a 16-fold increase in the expression of eNOS mRNA. The peak expression of inducible NOS (iNOS) in lung tissue specimens obtained from rats that received LPS was 45-fold higher than that in control rats. ET-1 is a potent vasoconstrictor and thereby may play an important role in the pathogenesis of acute lung injury in a septic rat model. The increased expression of NOS may result in excess NO production and may also play a role in the pulmonary complications of endotoxemia.     

Renin expression in the kidney and brain is reciprocally controlled by captopril

The tissue distribution of rat renin mRNA was examined. Sensitive RNase protection analyses demonstrated that renin mRNA are produced by the extra-renal tissues such as adrenal, brain, liver, lung, pituitary and testis. In response to sodium depletion and captopril treatment, the expression of mRNAs encoding rat renin were in a tissue-specific manner. The level of kidney renin mRNA remarkably increased in sodium-depleted rats treated with captopril, whereas that of brain renin mRNA definitely decreased. No significant change in the level of liver renin mRNA was observed after the same treatment. These results suggest that the expression of cerebral renin is regulated by physiological stimuli independent of its extra-cerebral expression.     

Alterations of gene expressions of preproET-1 and ET receptors in brains of endotoxemic Sprague-Dawley rats

During severe sepsis, several immunological defense mechanisms initiate a cascade of inflammatory events leading to multiorgan failure, including septic encephalopathy and ultimately death. Endothelin-1 (ET-1) has recently been investigated in different cerebral pathologies. Some reports suggest the involvement of ET-1 in sepsis. However, no study to date has reported the alterations in expression of the genes encoding preproET-1 and ET receptors in the frontal cortex of the septic brain. Male Sprague-Dawley (SD) rats 8 weeks of age were administered either saline or 15 mg/kg lipopolysaccharide (LPS) at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. The rats were sacrificed with ether, and the brain tissues were harvested. Systolic and diastolic blood pressure decreased 1 hr after LPS administration and then gradually returned to normal, without any change in the heart rate. We confirmed the induction of endotoxemia in the brains of SD rats by measuring the expression of nitric oxide synthase (NOS) mRNA induced in the cerebrum. The expression of inducible NOS (iNOS) mRNA in the brains of SD rat after LPS administration was 30-fold higher than that in the brains of control rats. mRNA expression of preproET-1 in the frontal cortex of SD rats after LPS administration was 2-fold higher than that in control rats. A time-dependent increase in the expression of the gene encoding the ET(A) receptor (vasoconstrictive property) after LPS administration was observed in SD rat brain, whereas expression of the gene encoding the ET(B) receptor (vasodilatatory property) showed an initial upregulation and then gradually decreased as sepsis progressed. In conclusion, we report for the first time that expressions of the genes encoding ET-1 and ET receptors are altered in the endotoxemic brain and that these alterations are time-dependent in SD rats. The alterations in the ET system in brain tissue observed in the present study may contribute to the understanding of the pathophysiological changes in the endotoxemic brain.     

Streptozocin-induced neuropathy is associated with altered expression of voltage-gated calcium channel subunit mRNAs in rat dorsal root ganglion neurones.

Voltage-gated calcium channels (VGCCs) within sensory neurones are believed to perform an important role in neuropathic pain. In the present study we examine the changes in VGCC mRNA which occur following streptozocin- (STZ) induced diabetic neuropathy using in situ hybridization. STZ caused a significant increase in alpha(2)delta(1), alpha(2)delta(2), and alpha(2)delta(3) mRNA levels in all neuronal cell types. Similarly, mRNA levels of alpha(1F), alpha(1I), and alpha(1S) were increased in all cell types studied whilst alpha(1A) and alpha(1G) mRNAs were specifically upregulated in medium and large diameter neurones. In conclusion, we demonstrate that the induction of diabetic neuropathy is associated with dramatic changes in the expression of VGCCs.     

Identification of adrenomedullin in avian type II pneumocytes: increased expression after exposure to air pollutants.

Adrenomedullin (AM) is a potent vasodilator peptide present in the lung of mammals where it is expressed mainly in the columnar epithelium and alveolar macrophages. AM increases the secretion of phosphatidylcholine by type II pneumocytes, which suggests a role as an autocrine modulator of surfactant secretion. In this study we show the expression of an AM-like protein in the lung of the pigeon, Columba livia. Using an antibody against its human ortholog, AM-like immunoreactivity was found to be associated with membranous structures of the multivesicular bodies of type II pneumocytes. We also studied the differential expression of AM-like peptide in the lung of pigeons exposed to polluted city air vs cleaner countryside conditions and found that AM-like expression was higher in city animals. Similar results were obtained in an experimental study in which pigeons were exposed to increasing concentrations of a single pollutant, ozone. Taken together, our findings support the implication of AM in the response of type II pneumocytes to air pollutants.     

Developmental expression of hexokinase 1 and 3 in rats

 Mammalian hexokinase types one and three (HK1 and HK3) are 100 kDa isozymes that phosphorylate glucose to glucose-6-phosphate. HK1 is present in most tissues but is especially prominent in brain and kidney. HK3 is less well studied, but may be most prominent in the spleen and lymphocytes. In this study, we determined the ontogeny of the expression of these isoforms in the rat. Using immunohistochemistry, we identified HK1 and HK3 immunoreactivity in the brain, heart, kidney, liver, skeletal muscle and spleen from gestational day 14 (E14) to 45 days after birth (P45). With the exception of the liver and spleen, we observed a similar age- and cell-dependent staining pattern for both isoforms in all organs studied. The brain and spleen were analyzed in more detail to identify specific regions of immunoreactivity during maturation. A transient expression of HK1 and HK3 was noted in the cell bodies of mature neurons, including layers V and VI of the cerebral cortex and the cerebellar Purkinje cells followed by localization to the white matter of the cerebrum and cerebellum. In the spleen, HK3 immunoreactivity was detected postnatally and appeared to track with the infiltration of B cells. Our demonstration of changing patterns of immunoreactivity for HK1 and HK3 in fetal and postnatal organs suggests that these HK isoforms are involved the process of development. We speculate that HK1 and HK3 share a complex interaction during development of these organs and regulate glucose metabolism at multiple levels during development. Accepted: 16 May 1997     

Detection of sarin in plasma of rats after inhalation intoxication

Abstract

Presently used methods for detection and diagnosis of the severity of intoxication with organophosphorus (OP) compounds are mostly those that quantify inhibition of blood cholinesterases. It was found that when plasma inhibited with OP compounds is incubated in the presence of a high concentration of fluoride ions, the organophosphate is released from the enzyme thus yielding a phosphofluoridate, which can be analyzed by gas chromatography and NP detection. In our study, the concentration of sarin released after fluoride ions were added to the plasma of sarin-poisoned rats was determined. Sarin amounts in plasma measured after refluoridation and plasma butyrylcholinesterase activity in ten rats, that were exposed to sarin vapors at concentration of 1.25 μg/L (E1 group) and 2.5 μg/L (E2 group) respectively, for 60 min. In the E2 group the concentration of sarin in plasma was nearly 2-fold higher than in the E1 group. These results correspond well with the concentrations of sarin vapors to which the animals were exposed. Both experimental groups of animals showed significant decreases in butyrylcholinesterase activity by more than 30%–36.4% (E1 group) and 47.0% (E2 group). The method of fluoride-induced reactivation provides a very good marker for monitoring sarin intoxication in laboratory animals determined previously mostly by ChE determination which does not allow any information on sarin amounts in plasma.     

Tissue-specific expression of PPAR mRNAs in diabetic rats and divergent effects of cilostazol

Cilostazol and ligands of peroxisome proliferator-activated receptors (PPARs) have been effectively used to alleviate diabetic complications, but the common and tissue-specific expression patterns of PPARs in different tissues in diabetic patients and those treated with cilostazol have not been reported. Here, we aimed to assess the effects of diabetes and cilostazol on mRNA expression of PPARalpha and PPARgamma in the aorta, renal cortex, and retina of diabetic rats treated with cilostazol for 8 weeks. PPARalpha mRNA expression showed uniform downregulation in all these tissues in diabetic rats, and this effect was reversed by cilostazol treatment. Surprisingly, PPARgamma mRNA expression was reduced in the renal cortex and retina, yet increased in the aorta of diabetic rats, although cilostazol still reversed these changes. Interestingly, cilostazol, a well-known phosphodiesterase 3 inhibitor and cAMP elevator, augmented cAMP content only in the aorta, but showed no significant effects in the renal cortex of diabetic rats. In conclusion, mRNA expression of PPARs is tissue-specific in diabetes and may be differently affected by cilostazol, possibly because of its tissue-specific effects on cAMP content.     

The age-related paraoxonase 1 response is altered by long-term caloric restriction in male and female rats

Caloric restriction (CR) has been shown to attenuate age-related oxidative damage and to improve major atherosclerotic risk factors. Paraoxonase 1 (PON1), an enzyme specifically associated with HDL containing apolipoproteins A-I and J, has been reported to prevent the proatherosclerotic effects of oxidized LDL. The aim of this study was to evaluate whether modulation of PON1 activity is part of the underlying CR mechanisms that attenuate the age-associated negative effects. Experimental groups were 1 year old rats of both genders subjected to 40% CR for 1 year and two ad libitum-fed groups, also including rats of both genders, euthanized at 6 months or 2 years. Aging impaired the serum lipid profile and increased lipid peroxidation, PON1 activities, and the content of both PON1 and apolipoprotein J in HDL, which suggests an HDL subfraction redistribution to protect LDL more effectively from oxidation. The CR-associated improved lipid profile and the decreased lipid peroxide levels would lead to the decreased arylesterase activity seen in old CR animals, suggesting that PON1 modulation is not an integral part of the main antioxidant mechanisms of CR but rather that CR would determine a more youthful and less oxidative situation in which the protection of LDL would be less necessary.     

Detection of sarin in plasma of rats after inhalation intoxication

Presently used methods for detection and diagnosis of the severity of intoxication with organophosphorus (OP) compounds are mostly those that quantify inhibition of blood cholinesterases. It was found that when plasma inhibited with OP compounds is incubated in the presence of a high concentration of fluoride ions, the organophosphate is released from the enzyme thus yielding a phosphofluoridate, which can be analyzed by gas chromatography and NP detection. In our study, the concentration of sarin released after fluoride ions were added to the plasma of sarin-poisoned rats was determined. Sarin amounts in plasma measured after refluoridation and plasma butyrylcholinesterase activity in ten rats, that were exposed to sarin vapors at concentration of 1.25 microg/L (E1 group) and 2.5 microg/L (E2 group) respectively, for 60 min. In the E2 group the concentration of sarin in plasma was nearly 2-fold higher than in the E1 group. These results correspond well with the concentrations of sarin vapors to which the animals were exposed. Both experimental groups of animals showed significant decreases in butyrylcholinesterase activity by more than 30%-36.4% (E1 group) and 47.0% (E2 group). The method of fluoride-induced reactivation provides a very good marker for monitoring sarin intoxication in laboratory animals determined previously mostly by ChE determination which does not allow any information on sarin amounts in plasma.     

Interaction between endothelial heme oxygenase-2 and endothelin-1 in altered aortic reactivity after hypoxia in rats

The aim of this study was to determine whether increased expression of heme oxygenase (HO) contributes to impairment of aortic contractile responses after hypoxia through effects on reactivity to endothelin-1 (ET-1). Thoracic aortas from normoxic rats and rats exposed to hypoxia (10% O2) for 16 or 48 h were mounted in organ bath myographs for contractile studies, fixed in paraformaldehyde, or frozen in liquid nitrogen for protein extraction. In rings from normoxic rats, the HO inhibitor tin protoporphyrin IX (SnPP IX, 10 microM) did not alter the response to phenylephrine or ET-1. In rings from rats exposed to 16-h hypoxia, maximum tension generated in response to these agonists was higher in endothelium-intact but not -denuded rings in the presence of SnPP IX. In rings from rats exposed to 48-h hypoxia SnPP IX increased contraction in endothelium-intact but not -denuded rings. In endothelium-intact aortic rings from rats exposed to 16-h hypoxia incubated with endothelin A receptor-specific antagonist BQ-123 (10(-7) M), SnPP IX did not alter phenylephrine-induced contraction. Aortic ET-1 protein levels, measured by radioimmunoassay, were increased in rats exposed to hypoxia for 16 and 48 h. Western blotting showed that HO-1 and HO-2 protein were increased after 16 h of hypoxia and returned to near-control levels after 48 h. Increase in HO-1 protein was detected in endothelium-intact and -denuded rings. Removal of endothelium abolished the increase in HO-2 immunoreactivity. Immunohistochemistry localized expression of HO-1 protein to vascular smooth muscle, whereas HO-2 was only detected in endothelium. HO-2 is expressed by aortic endothelial cells early during hypoxic exposure and impairs ET-1-mediated potentiation of contraction to alpha-adrenoceptor stimulation.     

Effects after inhalation of (1-->3)-beta-D-glucan in healthy humans

BACKGROUND AND AIM: This study was performed to assess the effects of an exposure to a pure (1-->3)-beta-D-glucan, a cell wall component of fungi, plants and certain bacteria. METHODS: Twenty-one healthy subjects inhaled saline or (1-->3)-beta-D-glucan suspended in saline in a random, double-blind, cross-over design. They were examined before exposure and 24 and 72h afterwards with spirometry, blood sampling and collection of induced sputum. Differential cell counts and eosinophilic cationic protein (ECP) were determined in blood and sputum, and myeloperoxidase (MPO), tumour necrosis factor-alpha (TNF-alpha), and interleukin (IL)-8 and IL-10 were determined in sputum supernatants. TNF-alpha was determined after cultivation of blood mononuclear cells. RESULTS: In sputum, inhalation of saline caused a significant increase in ECP and TNF-alpha. (1-->3)-beta-D-Glucan inhalation caused a further increase in these cytokines, although not statistically significantly different from the increase induced by inhalation of saline alone. In blood, the number of eosinophils was significantly decreased 72 h after the challenge with (1-->3)-beta-D-glucan. This effect was not found after the inhalation of saline alone. TNF-alpha production from stimulated blood mononuclear cells was significantly decreased 72 h after the (1-->3)-beta-D-glucan inhalation as compared with the increase induced by saline inhalation. CONCLUSIONS: The results suggest that (1-->3)-beta-D-glucan causes a different type of response as compared with inflammatory agents such as bacterial endotoxin that cause a neutrophil-dominated inflammatory response.